Abstract

Secreted levels of HSP90α and overexpression of TCF12 have been associated with the enhancement of colorectal cancer (CRC) cell migration and invasion. In this study, we observed that CRC patients with tumor TCF12 overexpression exhibited both a higher rate of metastatic occurrence and a higher average serum HSP90α level compared with patients without TCF12 overexpression. Therefore, we studied the relationship between the actions of secreted HSP90α and TCF12. Like overexpressed TCF12, secreted HSP90α or recombinant HSP90α (rHSP90α) induced fibronectin expression and repressed E-cadherin, connexin-26, connexin-43, and gap junction levels in CRC cells. Consistently, rHSP90α stimulated invasive outgrowths of CRC cells from spherical structures during three-dimensional culture. rHSP90α also induced TCF12 expression in CRC cells. Its effects on CRC cell epithelial-mesenchymal transition, migration, and invasion were drastically prevented when TCF12 was knocked down. This suggests that TCF12 expression is required for secreted HSP90α to enhance CRC cell spreading. Through the cellular receptor CD91, rHSP90α facilitated the complex formation of CD91 with IκB kinases (IKKs) α and β and increased the levels of phosphorylated (active) IKKα/β and NF-κB. Use of an IKKα/β inhibitor or ectopic overexpression of dominant-negative IκBα efficiently repressed rHSP90α-induced TCF12 expression. Moreover, κB motifs were recognized in the gene sequence of the TCF12 promoter, and a physical association between NF-κB and the TCF12 promoter was detected in rHSP90α-treated CRC cells. Together, these results suggest that the CD91/IKK/NF-κB signaling cascade is involved in secreted HSP90α-induced TCF12 expression, leading to E-cadherin down-regulation and enhanced CRC cell migration/invasion.

Secreted HSP90α induces fibronectin expression but represses E-cadherin, connexin-43, and connexin-26 levels in CRC cells.A, RT-PCR was performed to detect E-cadherin, fibronectin, connexin-43, and connexin-26 mRNA levels in HCT-115 and SW480 cells treated for 24 h with serum starvation CM in the presence of preimmune IgG or anti-HSP90α antibody. Representative data from three independent experiments are shown. The quantitative data were also obtained using real-time RT-PCR. Taking HCT-115 cells as an example, cellular E-cadherin, connexin-26, and connexin-43 mRNA levels were decreased to 13, 11, and 14%, respectively; however, fibronectin expression was increased to 790% in response to CM plus preimmune IgG. The levels of E-cadherin, connexin-26, connexin-43, and fibronectin mRNA expression could be recovered to 50, 91, 79, and 129%, respectively, when anti-HSP90α antibody was added instead, suggesting that the effects of CM were at least partly attributable to the action of HSP90α secreted in CM. CTRL, control. B, immunoblot analyses were performed to detect E-cadherin, fibronectin, connexin-43, and connexin-26 protein levels in HCT-115 and SW480 cells treated for 24 h with CM in the absence or presence of preimmune IgG or anti-HSP90α antibody. Representative data from three independent experiments are shown. C, RT-PCR was performed to analyze E-cadherin, fibronectin, connexin-43, and connexin-26 mRNA levels in HCT-115, LoVo, SW480, and SW620 cells treated for 24 h with 15 μg/ml rHSP90α. Representative images from three independent experiments are shown. The quantitative data were also obtained using real-time RT-PCR. Taking HCT-115 cells as an example, cellular levels of E-cadherin, connexin-26, and connexin-43 mRNAs were decreased to 9, 24, and 28%, respectively; however, fibronectin expression was increased by 4.2-fold in rHSP90α-treated cells compared with control cells treated with PBS. D, immunoblot analyses were performed to detect E-cadherin, fibronectin, connexin-43, and connexin-26 protein levels in HCT-115, LoVo, SW480, and SW620 cells treated for 24 h with PBS or 15 μg/ml rHSP90α. Representative data from three independent experiments are shown.

rHSP90α inhibits gap junction activity in CRC cells and induces invasive outgrowths of CRC cells from spherical structures during three-dimensional culture.A and B, the Calcein transfer assay was performed to evaluate the effect of rHSP90α on CRC cell gap junction activity. HCT-115 and SW480 cells after 24-h PBS or rHSP90α treatment were labeled with Calcein acetoxymethyl ester (Calcein) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) dyes and then added to a monolayer of unstained untreated cells of the same type for 0.2 or 3 h of co-culture. Finally, the monolayer of cells was trypsinized and analyzed by flow cytometry. The dot plots are representative results obtained from HCT-115 cells (A). The cells in the R1 region were categorized as Calcein-accepting cells. The ratio of Calcein-accepting cells (designated as % Transfer) was quantified using CellQuest software, and the means ± S.D. of three independent experiments show that cellular gap junction activity was significantly inhibited after rHSP90α treatment (p < 0.05; B). C, rHSP90α represses the three-dimensional spherical structures assembled by CRC cells. HCT-115 and SW480 cells were cultivated in 2% Matrigel-supplemented medium until spherical structures formed. These cells were then treated with 15 μg/ml rHSP90α for 72 h. The change in morphology was observed using an Olympus IX71 inverted microscope.

rHSP90α enhances CRC cell migration and invasion.A, rHSP90α increases the migration activities of HCT-115 and SW480 cells. Cell migration tracks of rHSP90α-treated or PBS-treated control cells were monitored for 16 h by time-lapse photography and analyzed using Image-Pro Plus software (upper panels). Twenty cells in each treatment group were randomly selected, and their accumulated and oriented migration distances were quantified and expressed as means ± S.D. (lower panels). The results shown are representative of three independent experiments and indicate that cell activity was significantly enhanced by rHSP90α treatment (p < 0.05). B, rHSP90α increases cell invasiveness in HCT-115 and SW480 cells. HCT-115 and SW480 cells were pretreated for 24 h with PBS or rHSP90α and allowed to invade through Matrigel for 16 h. Invasive cells on the filters of the Transwell inserts were counted using Image-Pro Plus software. The means ± S.D. of three independent experiments show that cell invasion activity was significantly enhanced by rHSP90α treatment (p < 0.05). C, RT-PCR and immunoblot analyses were performed to show the induction of mRNA and protein levels of integrin αV, MMP-9, and MMP-2 in HCT-115, LoVo, SW480, and SW620 cells treated for 24 h with 15 μg/ml rHSP90α. Representative data from three independent experiments are shown.

TCF12 is required for rHSP90α-induced E-cadherin down-regulation.A, rHSP90α induces TCF12 expression in CRC cells. RT-PCR and immunoblot analyses were performed to demonstrate that TCF12 mRNA and protein levels were both increased in 15 μg/ml rHSP90α-treated HCT-115, LoVo, SW480, and SW620 cells. Representative images from three independent experiments are shown. B and C, TCF12 knockdown prevents rHSP90α-induced E-cadherin down-regulation. The LoVo and SW620 cell clones stably expressing control shRNA or TCF12 shRNA (sequence 1 or 2) were treated with PBS or rHSP90α. RT-PCR and immunoblot analyses were performed to investigate TCF12 and E-cadherin expression levels. Representative images from three independent experiments are shown in B. TCF12 shRNA sequences 1 and 2 were against nucleotides 986∼1006 and 1727∼1747 of TCF12 mRNA, respectively. The sequence of the control shRNA used did not match any known human gene. Real-time RT-PCR was also performed, and the means ± S.D. of three independent experiments are expressed in C to show relative mRNA levels of TCF12, Twist, E-cadherin, and fibronectin in the representative cell clones (stably expressing control shRNA or TCF12 shRNA sequence 1) after treatment with or without rHSP90α.

TCF12 is required for rHSP90α to inhibit CRC cell gap junction activity and to induce invasive outgrowths of CRC cells from three-dimensional spherical structures.A and B, the Calcein assay was performed to evaluate the gap junction activities in representative control (ctrl) and TCF12-knockdown LoVo (A) and SW620 (B) cells after treatment with PBS or 15 μg/ml rHSP90α for 24 h. Cellular gap junction activity was evaluated by quantifying the ratio of Calcein-accepting cells. The means ± S.D. of three independent experiments revealed that cellular gap junction activity was significantly inhibited by rHSP90α treatment (p < 0.05) only in control (but not TCF12-knockdown) LoVo and SW620 cells, suggesting that induction of TCF12 expression is required for rHSP90α-induced inhibition of cellular gap junction activity. C and D, cell invasive outgrowths from three-dimensional spherical structures were significantly induced by rHSP90α only in control (but not TCF12-knockdown) LoVo (C) and SW620 (D) cells. Cells were cultivated in a three-dimensional culture manner as described above and treated with 15 μg/ml rHSP90α for 72 h. The results suggest that TCF12 expression is required for rHSP90α to repress cell-cell junctions and spherical structures in CRC cells.

TCF12 is involved in rHSP90α-induced CRC cell migration and invasion. Cell migration tracks were monitored and analyzed for representative control (ctrl) and TCF12-knockdown LoVo (A) and SW620 (B) cells during incubation with PBS or 15 μg/ml rHSP90α for 16 h. Twenty cells in each treatment group were randomly selected, and their accumulated and oriented migration distances were quantified and expressed as means ± S.D. The representative results of three independent experiments indicate that rHSP90α-induced CRC cell migration could be significantly prevented by knockdown of cellular TCF12 expression. *, p < 0.05. Additionally, cell invasiveness was investigated by the Transwell invasion assay for control and TCF12-knockdown LoVo (C) and SW620 (D) cells that had been treated for 24 h with PBS or 15 μg/ml rHSP90α. The means ± S.D. of three independent experiments show that rHSP90α-induced CRC cell invasion could be significantly prevented by knockdown of cellular TCF12 expression.

rHSP90α induces cellular TCF12 expression through the NF-κB-dependent pathway.A, RT-PCR was performed to investigate TCF12 mRNA levels in HCT-115 and SW480 cells treated for 24 h with 15 μg/ml rHSP90α in the presence of inhibitors of MEKK (MEKKi; PD98059, 5 μm), JNK (JNKi; SP600125, 5 μm), p38 MAPK (p38i; SB202190, 5 μm), and IKKα/β (IKKi; 6-amino-4-(4-phenoxyphenylethylamino)quinazoline, 0.1 μm). Representative results from three independent experiments are shown. The data indicate that the IKKα/β inhibitor, but not the others, can drastically abolish rHSP90α-induced TCF12 mRNA expression. DMSO, dimethyl sulfoxide. B, immunoblot analyses of the phosphorylation status of IKKα/β and NF-κB in HCT-115 and SW480 cells treated for 24 h with rHSP90α in the absence or presence of the IKKα/β inhibitor. Representative results from three independent experiments are shown. The data confirm that the IKKα/β inhibitor indeed repressed rHSP90α-induced IKKα/β and NF-κB phosphorylation. C, HCT-115 and SW480 cells were transfected for 48 h with a control vector or dnIκBα-overexpressing plasmid (pRc/CMV-IκB72). Transfected cells were harvested and further treated with PBS or 15 μg/ml rHSP90α for another 24 h. Nuclear extracts were prepared for immunoblot analyses of NF-κB and TCF12 levels. The results show that dnIκBα was efficient in inhibiting rHSP90α-induced nuclear levels of NF-κB and TCF12, confirming that NF-κB is involved in rHSP90α-induced TCF12 expression. The levels of proliferating cell nuclear antigen (PCNA) were used as internal controls. D, the ChIP assay was performed to indicate a physical association of NF-κB with the TCF12 gene promoter in HCT-115 and SW480 cells treated with 15 μg/ml rHSP90α for 24 h. Representative results from three independent experiments are shown.

rHSP90α induces the NF-κB signaling pathway through the CD91 receptor.A, immunoblot analyses of the phosphorylation status of IKKα/β and NF-κB in HCT-115 and SW480 cells treated for 24 h with 15 μg/ml rHSP90α in the presence of the antagonizing antibody against CD91. Representative results from three independent experiments indicate that rHSP90α-induced IKKα/β and NF-κB phosphorylation could be prevented by anti-CD91 antibody, suggesting that rHSP90α induces IKK signaling through CD91. B, rHSP90α induces a physical association of CD91 with IKKα and IKKβ. HCT-115 and SW480 cells were treated for 24 h with 15 μg/ml rHSP90α and then double-stained with anti-CD91 and anti-IKKα antibodies, with anti-CD91 and anti-IKKβ antibodies, or with anti-IKKα and anti-IKKβ antibodies, followed by the proximity ligation assay. After nuclei were counterstained with DAPI, red fluorescent dots, resulting from the direct contacts of CD91 with IKKα/β and of IKKα with IKKβ, were observed by confocal microscopy.